scholarly journals The α-5 segment of Bacillus thuringiensis δ-endotoxin: in vitro activity, ion channel formation and molecular modelling

1994 ◽  
Vol 304 (3) ◽  
pp. 895-902 ◽  
Author(s):  
E Gazit ◽  
D Bach ◽  
I D Kerr ◽  
M S P Sansom ◽  
N Chejanovsky ◽  
...  
Keyword(s):  
Ion Channels ◽  
Bacillus Thuringiensis ◽  
Molecular Modelling ◽  
Lipid Membranes ◽  
Functional Characterization ◽  
Coiled Coil ◽  
Emission Spectra ◽  
Blue Shift ◽  
Delta Endotoxin

A peptide with a sequence corresponding to the highly conserved alpha-5 segment of the Cry delta-endotoxin family (amino acids 193-215 of Bacillus thuringiensis CryIIIA [Gazit and Shai (1993) Biochemistry 32, 3429-3436]), was investigated with respect to its interaction with insect membranes, cytotoxicity in vitro towards Spodoptera frugiperda (Sf-9) cells, and its propensity to form ion channels in planar lipid membranes (PLMs). Selectively labelled analogues of alpha-5 at either the N-terminal amino acid or the epsilon-amine of its lysine, were used to monitor the interaction of the peptides with insect membranes. The fluorescent emission spectra of the 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labelled alpha-5 peptides displayed a blue shift upon binding to insect (Spodoptera littoralis) mid-gut membranes, reflecting the relocation of the fluorescent probes to an environment of increased apolarity, i.e. within the lipidic constituent of the membrane. Moreover, midgut membrane-bound NBD-labelled alpha-5 peptides were protected from enzymic proteolysis. Functional characterization of alpha-5 has revealed that it is cytotoxic to Sf-9 insect cells, and that it forms ion channels in PLMs with conductances ranging from 30 to 1000 pS. A proline-substituted analogue of alpha-5 is less cytolytic and slightly more exposed to enzymic digestion. Molecular modelling utilizing simulated annealing via molecular dynamics suggests that a transbilayer pore may be formed by alpha-5 monomers that assemble to form a left-handed coiled coil of approximately parallel helices. These findings further support a role for alpha-5 in the toxic mechanism of delta-endotoxins, and assign alpha-5 as one of the transmembrane helices which form the toxic pore. The suggested role is consistent with the recent finding that cleavage of CryIVB delta-endotoxin in a loop between alpha-5 and alpha-6 is highly important for its larvicidal activity [Angsuthanasombat, Crickmore and Ellar (1993) FEMS Microbiol. Lett. 111, 255-262].

scholarly journals Effects of Bacillus thuringiensis δ-Endotoxin on Insect and Mammalian Cells In Vitro

1980 ◽  
Vol 15 (2) ◽  
pp. 133-139 ◽  
Author(s):  
Junko NISHITSUTSUJI-UWO ◽  
Yasuhisa ENDO ◽  
Michio HIMENO
Keyword(s):  
Bacillus Thuringiensis ◽  
Mammalian Cells ◽  
Delta Endotoxin

scholarly journals The C-terminal region of the plasmid partitioning protein TubY is a tetramer that can bind membranes and DNA

2020 ◽  
Vol 295 (51) ◽  
pp. 17770-17780
Author(s):  
Ikuko Hayashi
Keyword(s):  
Lipid Membranes ◽  
Binding Protein ◽  
Coiled Coil ◽  
Type Iii ◽  
Daughter Cells ◽  
Lysine Residues ◽  
Stable Maintenance ◽  
Biochemical Analyses

Bacterial low-copy-number plasmids require partition (par) systems to ensure their stable inheritance by daughter cells. In general, these systems consist of three components: a centromeric DNA sequence, a centromere-binding protein and a nucleotide hydrolase that polymerizes and functions as a motor. Type III systems, however, segregate plasmids using three proteins: the FtsZ/tubulin-like GTPase TubZ, the centromere-binding protein TubR and the MerR-like transcriptional regulator TubY. Although the TubZ filament is sufficient to transport the TubR-centromere complex in vitro, TubY is still necessary for the stable maintenance of the plasmid. TubY contains an N-terminal DNA-binding helix-turn-helix motif and a C-terminal coiled-coil followed by a cluster of lysine residues. This study determined the crystal structure of the C-terminal domain of TubY from the Bacillus cereus pXO1-like plasmid and showed that it forms a tetrameric parallel four-helix bundle that differs from the typical MerR family proteins with a dimeric anti-parallel coiled-coil. Biochemical analyses revealed that the C-terminal tail with the conserved lysine cluster helps TubY to stably associate with the TubR-centromere complex as well as to nonspecifically bind DNA. Furthermore, this C-terminal tail forms an amphipathic helix in the presence of lipids but must oligomerize to localize the protein to the membrane in vivo. Taken together, these data suggest that TubY is a component of the nucleoprotein complex within the partitioning machinery, and that lipid membranes act as mediators of type III systems.

scholarly journals 2066 Functional characterization of mutant BRCA1

2018 ◽  
Vol 2 (S1) ◽  
pp. 13-13
Author(s):  
John Barrows ◽  
David Long
Keyword(s):  
Functional Characterization ◽  
Coiled Coil ◽  
Wild Type ◽  
Coiled Coil Region ◽  
Egg Extracts ◽  
Study Population ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

OBJECTIVES/SPECIFIC AIMS: The objective of this work is to determine the mechanistic consequences of BRCA1 mutants in inter-strand crosslink (ICL) repair. METHODS/STUDY POPULATION: Our lab uses Xenopus egg extracts to study ICL repair. These extracts can be depleted of endogenous BRCA1 by immunoprecipitation. The goal of this work is to rescue endogenous depletion with in vitro translated, wild type BRCA1. Once achieved, we can supplement the depleted extract with BRCA1 mutants to access their function in ICL repair. RESULTS/ANTICIPATED RESULTS: We hypothesize that the BRCT and RING domain mutations will abrogate ICL repair, while mutations in the coiled coil region will not affect repair. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings will have an immense impact on the understanding of BRCA1 domains. Importantly these results will spur personalized therapy of BRCA1 mutants by showing which domains are sensitive to cross-linking agents.

scholarly journals Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2

2009 ◽  
Vol 191 (22) ◽  
pp. 6843-6854 ◽  
Author(s):  
Tsuyoshi Miki ◽  
Yoshio Shibagaki ◽  
Hirofumi Danbara ◽  
Nobuhiko Okada
Keyword(s):  
Protein Interactions ◽  
Type Iii Secretion ◽  
Functional Characterization ◽  
Coiled Coil ◽  
Type Iii Secretion System ◽  
Pathogenicity Island ◽  
Secretion System ◽  
Type Iii ◽  
Serovar Typhimurium

ABSTRACT The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An S. enterica serovar Typhimurium ssaE mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an sseA deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast, ssaE mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an ssaE(I55G) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for Salmonella virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery.

Bacillus thuringiensis var israelensis crystal delta-endotoxin: effects on insect and mammalian cells in vitro and in vivo

1983 ◽  
Vol 60 (1) ◽  
pp. 181-197 ◽  
Author(s):  
W.E. Thomas ◽  
D.J. Ellar
Keyword(s):  
Body Weight ◽  
Bacillus Thuringiensis ◽  
Mammalian Cells ◽  
Choristoneura Fumiferana ◽  
Similar Response ◽  
Parasporal Crystal ◽  
Discontinuous Sucrose Gradient ◽  
Delta Endotoxin

Bacillus thuringiensis var israelensis parasporal crystal delta-endotoxin was purified by ultracentrifugation on a discontinuous sucrose gradient. Native delta-endotoxin crystals showed no detectable toxicity in the vitro and in vivo systems that are described. By contrast alkali-solubilized crystal delta-endotoxin caused rapid cytological and cytopathological changes in Aedes albopictus, Choristoneura fumiferana 63 CF1, Spodoptera frugiperda and Trichoplusia ni cell lines as observed by phase-contrast microscopy and vital staining. Mouse fibroblasts, primary pig lymphocytes and three mouse epithelial carcinoma cell types showed a similar response to the alkali-soluble crystal delta-endotoxin. In addition the soluble crystal delta-endotoxin protein caused haemolysis of rat, mouse, sheep, horse and human erythrocytes. Intravenous administration of the alkali-soluble crystal delta-endotoxin to Balb. c mice at a dose rate of 15–30 micrograms of protein per gram body weight resulted in rapid paralysis followed by death within 12h. Subcutaneous inoculation of 15–30 micrograms of protein per gram body weight resulted in death of suckling mice in 2–3 h. The alkali-solubilized crystal delta-endotoxin was not toxic however, when administered per os. A comparison is made with a similar alkali-soluble fraction from the parasporal crystal delta-endotoxin of B. thuringiensis var kurstaki. With the exception of the Lepidopteran cell line, Choristoneura fumiferana 63 CF1, this soluble crystal delta-endotoxin protein showed no in vitro or in vivo toxicity, and no haemolytic activity.

Characterization and partial purification of a plasma membrane receptor for Bacillus thuringiensis var. kurstaki lepidopteran-specific delta-endotoxin

1986 ◽  
Vol 83 (1) ◽  
pp. 89-101
Author(s):  
B.H. Knowles ◽  
D.J. Ellar
Keyword(s):  
Plasma Membrane ◽  
Bacillus Thuringiensis ◽  
Choristoneura Fumiferana ◽  
Galactose Oxidase ◽  
Partial Purification ◽  
Wide Range ◽  
Delta Endotoxin ◽  
Plasma Membrane Receptor ◽  
Insect Gut

The lepidopteran-specific P1 delta-endotoxin of Bacillus thuringiensis var. kurstaki HD-1 was activated in vitro using insect gut proteases and found to be highly specific for the lepidopteran cell line Choristoneura fumiferana CF1 among a wide range of lepidopteran and dipteran cell lines tested. The toxicity of P1 against CF1 cells is inhibited by N-acetylgalactosamine (GalNAc), and the lectins soybean agglutinin (SBA) and wheat-germ agglutinin. Protein blotting was used to identify a glycoprotein of 146 X 10(3) Mr in the plasma membrane of CF1 cells, capable of binding both the toxin and SBA, which is specific for GalNAc. This glycoprotein was labelled using galactose oxidase and sodium boro-[3H]hydride and solubilized in Triton X-100 before partial purification by affinity chromatography on SBA-agarose. We propose that this glycoprotein is a good candidate for the cellular receptor of the lepidopteran-specific P1 delta-endotoxin of B. thuringiensis var. kurstaki HD-1.

scholarly journals Mutagenesis of two surface-exposed loops of the Bacillus thuringiensis CryIC δ-endotoxin affects insecticidal specificity

1994 ◽  
Vol 302 (2) ◽  
pp. 611-616 ◽  
Author(s):  
G P Smith ◽  
D J Ellar
Keyword(s):  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Time Dependence ◽  
Time Course ◽  
Site Directed Mutagenesis ◽  
Sf9 Cells ◽  
Degenerate Oligonucleotide ◽  
Delta Endotoxin ◽  
A Cell

Site-directed mutagenesis was used to determine the role of two surface-exposed loops (Gly-317-Phe-320 and Gln-374-Pro-377) in the insecticidal specificity of the Bacillus thuringiensis CryIC delta-endotoxin. Mutant toxins were generated by PCR using degenerate oligonucleotide primers, and expressed in Escherichia coli. More than 50 mutant toxins were screened for toxicity to the lepidopteran Spodoptera frugiperda Sf9 cell line using an in vitro lawn assay. A panel of these mutant toxins, which included toxic and non-toxic variants from both loops, was further screened for activity towards Aedes aegypti larvae. The activity of these mutants to Sf9 cells was quantified more precisely using a cell lysis assay. Three categories of mutants were identified: (1) those non-toxic to either Sf9 cells or Aedes aegypti larvae; (2) those fully toxic to both genera; and (3) those which were only toxic to Sf9 cells. For the first loop, the differential specificity was not restricted to any single residue. In the second loop, two mutant toxins with a Pro-377-->Ala substitution displayed this phenotype. The time dependence of toxicity towards Sf9 cells was examined using the same panel of mutants. All toxic mutants displayed an identical time course to the wild-type toxin, with the exception of the two Pro-377-->Ala mutants of the second loop. These toxins displayed a lower time dependence, no cell death occurring within the first hour of incubation. These results show that the two loops are important determinants of both the activity and specificity of the CryIC delta-endotoxin.

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